The invention is suitable for the detection of the yeast species described herein from theoretically any kind of sample, preferably from a foodstuff, a food processing intermediate or a food raw material.
The selective detection and separation of yeast species having a role in beer and wine production that are in many cases detrimental, from wild yeast species is of particular significance.
Loureiro V and Malfeito-Ferreira M. [Spoilage yeasts in the wine industry. Int J Food Microbiol. 2003; 86(1-2):23-50.] give a detailed summary about microbiological problems caused by yeasts that are present in wine. The authors of the publication talk about methods that are presently known for detecting yeasts that cause deterioration in food, and analyze factors that help the colonization of yeasts on grapes and in wine.
Depreciation of wine that are originated in microbiological reasons cause a serious loss worldwide, affecting wine in premium categories, that are being matured in a wood barrel, even harder.
As this problem has a huge economic significance, procedures that recognize the presence and multiplication of harmful microorganisms on time are extremely important. With the help of these, the infestation can be prevented, or the intervention to lower the extent of deterioration can be done targetedly.
The majority of microorganisms that have a role in the creation of wine get into the process from the grapes or the wine-making equipment.
The species composition and the number of cells of the microflora change depending on the circumstances, the number of bacteria is infinitesimal, yeasts dominate. Among the yeasts, apart from the cultured (noble) strain of Saccharomyces there are many wild yeasts present, that dominate the fermenting medium for a long time. Among the wild yeasts, the genera of Brettanomyces/Dekkera and the Zygosaccharomyces tolerate high level of alcohol concentration relatively well and are resistant of the usual wine making procedures, therefore if they proliferate during the maturation of the wine it can cause the deterioration of it.
The genera of Zygosaccharomyces, especially the Zygosaccharomyces bailii can generate undesirable secondary fermentation and the formation of an adverse aroma in wines with residual sugar content, but at the same time they have a very important role in commencing the fermentation of high sugar content must and in fermenting must with inappropriate glucose: fructose proportion.
There is an adverse phenol-like character (the so called “brettyness”) that is caused by the metaboism byproduct of the species of Brettanomyces (mainly the B. bruxellensis) particularly during the production of red wine and as a consequence there is a significant decrease in quality and price. These non desirable components of flavor were described by sensory evaluations as “disinfectant”, “bretty”, “leather”, “wet dog”, “rancid”, “sweaty horse”, “ordure”, “stall” and “animal character”. The compounds arisen by the influence of Brettanomyces, in small quantities can contribute to the complexity of the wine (mild animality), however, above a certain number of cells the presence of the Brettanomyce is non-desirable. Their proliferation at an early stage of maturation can be prevented with carbon monoxide treatment, to which these yeasts are relatively sensitive. Although the sulphurization itself can influence the smell of the wine in a disadvantageous way and suppress the development, therefore the treatment has to be targeted. The condition of targeted intervention is the early identification of the presence of Brettanomyces in the maturing wine, when the number of these microorganisms and the concentration of their metabolism products are still low.
For this, we can use a number of analytical methods (ELISA, molecular biology methods, flow cytometry, culturing, chromatography methods), but most of them require special equipment and knowledge, furthermore they are very time consuming and sumptous, because in the medium there are a large number of other microorganisms that makes demonstrability very difficult.
For example, Cocolin and his co-workers [Molecular Detection and Identification of Brettanomyces/Dekkera bruxellensis and Brettanomyces/Dekkera anomalus in Spoiled Wines. Appl Environ Microbiol. 2004; 70(3): 1347-1355.] created a PRC-RFLP test for the identification of Brettanomyces bruxellensis and Brettanomyces anomalus. The key of their method is that different patterns for the two species are obtained, when a DNA fragment that was propagated during the PCR reaction is digested with restriction enzyme. The method is highly sensitive, and is suitable for detecting and separating two Brettanomyce species, however, it requires special lab equipment and it is relatively costly.
Phister and Mills Meal-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine. Applied and Environmental Microbiology. 2003; 69(147430-74341 used the RT-PCR method for identifying the Dekkera (Brettanomyces) bruxellensis in wine. The method is very sensitive (it is able to make a detection in a concentration of 1 cell/ml) and selective, but it is even more expensive than the traditional PCR, the equipment costs are also high and its use requires appropriately qualified personnel.
Stender and his colleagues [Identification of Dekkera bruxellensis (Brerranomyces) from wine by fluorescence in situ hybridization using peptide nucleic acid probes. Appl. Environ. Microbiol. 67:938-941 (2001)] detected Dekkera (Brettanomyces) bruxellensis from wine, Connell and his colleagues [Rapid Detection and Identification of Brettanomyces from Winery Air Samples Based on Peptide Nucleic Acid Analysis. Am. J. Enol. Vitic. 2002; 53(4): 322-24] detected Brettanomyces strains from the air of cellars with chemiluminescence in situ hybridization method (with the application of peptide nucleic acid probe). The method is highly sensitive, but is based on culturing, therefore it is time consuming and requires qualified workers, additionally, the cost of the tests are significant.
Mitrakul and his colleagues [Discrimination of Brettanomyces/Dekkera yeast isolates from wine by using various DNA fingerprinting methods. Food Microbiol. 1999 16 3-14.] used the RAPD-PCR method for the identification of Brettanomyces/Dekkera strains. This method is suitable for species and strain identification, but its condition is having a special instrument (PCR). The authors used it in combination with other identification methods, which were based on culturing and physiological tests, therefore the assay also became time consuming.
Ibeas and his colleagues [Detection of Dekkera-Brettanomyces Strains in Sherry by a Nested PCR Method. Appl. Environ. Microbiol. 1996, 62(3) 998-1003] identified Brettanomyces/Dekkera strains in sherry, with “nested” PCR. This method is highly sensitive, it does not require growing the strains, therefore it gives a quick, reliable result in 10 hours. Although it requires special equipment, and remains of sherry in the sample block the reaction, thus we can get a false result.
The Oeno Yeast Kit (Partec) identification is a fluorescent detection method, based on flow cytometry, that detects metabolically active yeast cells. Similarly to other cytometric procedures, this method is not specific to Brettanomyces/Dekkera species, therefore it can only be used in wine samples that are in the phase of maturing, when the presence of other yeasts are not probable. The test is expensive and needs special instruments.
To sum up, the advantage of molecular methods are selectivity and quickness, the advantage of instrumental methods are accuracy and quickness. Their disadvantage is that they require special and expensive instruments, the reaction is quite costly and the implementation and evaluation require qualified personnel. The common disadvantage of procedures of molecular biology is that if they identify one or two species, other species that can trigger deterioration of wine and food stay hidden.
The solution to this problem is using a medium, that is selective to the species of Brettanomyces/Dekkera and/or Zygosaccharomyces. The significant benefit of this medium is that using it does not require special equipment, neither microbiologist, nor analytical qualifications, and the examination can be carried out and evaluated in a wine cellar by an oenologist. A disadvantage of this technique that the selectivity of the medium is limited, therefore other microorganisms, e.g. colonies of wild yeast might appear (false positive result).
Renouf V. and Lonvaud-Funel A. [Development of an enrichment medium to detect Dekkera/Brettanomyces bruxellensis, a spoilage wine yeast, on the surface of grape berries. Microbiol Res. 2007; 162(2):154-67.] created a selective medium and with that, the species of Dekkera/Brettanomyces can be enriched, thus the procedure of indentification can be made sensitive.
Barata A. and his colleagues [Ascomycetous yeast species recovered from grapes damaged by honeydew and sour rot. Journal of Applied Microbiology, 2008 104(4) 1182-1191.] rose the alcohol content and used cycloheximide only as a source of carbon, thus assuring that their medium is selective for species of Dekkera/Brettanomyces. 
Schuller D. and his colleagues C. [A differential medium for the enumeration of the spoilage yeast Zygosaccharomyces bailii in wine. J Food Prot. 2000 63(11):1570-5.] created a medium that serves the selective culturing of Zygosaccharomyces bailii. With the proper adjustment of the concentration of formic acid and glucose they made the medium so selective, that only the Z. bailii caused a pH drift to alkaline direction, which resulted in a change of colour of the medium.
The aim of Hocking A D. [Media for preservative resistant yeasts: a collaborative study. Int J Food Microbiol. 1996 29(2-3):167-75] was creating a medium that is most suitable for identifying yeast that is resistant to preservatives in food. According to the publication, they authors examined 5 media, from which 3 were selective. Two out of 3 were made selective by adding acetic acid, while the third medium was a ZBM (Zygosaccharomyces bailii) medium, which contained tripan blue paint. When comparing the efficacy of the media, the ZBM medium appeared to be adequetly selective for Z. bailii, however, it was less suitable for counting because its growing inhibition effect.
By applying the above mentioned methods, media can be made selective for growing yeasts. An additional task is to identify the given yeast species, which in case of wild yeast is often done by scent sample, by adding a compound to the medium, e.g. p-coumaric acid, which transforms the yeast to a distinctive smelling compound.
Couto J. A. and his colleagues [A simple cultural method for the presumptive detection of the yeasts Brettanomyces/Dekkera in wines. Left Appl Microbiol. 2005 41(6):505-10.] presents an easy and reliable method in their publication for identifying the Brettanomyces/Dekkera yeasts. The base of their method is utilizing selective medium, which contains glucose, cycloheximide, chloramphenicol and p-coumaric acid. The presence of yeasts are evaluated by turbidity and scent.
Rodrigues N, and his colleagues [Development and use of a new medium to detect yeasts of the genera Dekkera/Brettanomyces. J Appl Microbiol. 2001 90(4):588-99.] developed a selective medium as well, for identifying species of Dekkera/Brettanomyces in an environment connected to wine making. They ensured the selectiveness of the medium by adding ethanol and cycloheximide. They proved the identification of acid producing strains by adding bromocresol green. Adding p-coumaric acid ensured the identification of Dekkera/Brettanomyces strains based on scent samples.
During the method applied by Lebrun Labs (Easy Blue Brettanomyces Test Kit), the selective medium that blocks the growth of most yeasts discolours from the effect of acids produced by microbes (pH change). Furthermore, the Dekkera/Brettanomyces colonies produce a distinctive smelling, volatile compound from the p-coumaric acid in the medium, whose perception happens by smelling it, so it requires experience and/or a comparative scent sample. Although this method does not require qualification or special instruments, it has its disadvantages: the medium is not absolute selective, which can lead to false positive results.
During the supplementary usage of p-coumaric acid, the metabolite it formulates [4-ethylfenol (4-EP), 4-ethylguaiacol (4-EG), isovaleric acid] has a smell that is typical of Brettanomyces/Dekkera yeasts, whose identification requires experience and/or scent sample. Taking a scent sample means opening the Petri dish again and again, which become a potential source of infection themselves.
Therefore, according to the technical knowledge, there were proposals for solutions, where the mentioned species of wild yeast were identified in a chromogenic medium, based on a reaction that was accompanied by discolouration.
Loureiro V, Malfeito-Ferreira M. [“Spoilage yeasts in the wine industry.” Int J Food Microbiol. 2003 86(1-2):23-50.] have a detailed, in-depth summary where they discuss the colony of yeasts on grapes and in wines, as well as mentioning the industrial identification techniques. They present the components that must be found in general media that are for identifying yeasts, and in relation to these they mention indicators that are used in these media: bromocresol green and bromphenol blue.
In the international publication no. WO0073494 (A1) Leao Cecilia and her colleagues describe media that are eligible for identifying species of Zygosaccharomyces, such as Zygosaccharomyces bailii and Zygosaccharomyces bisporus, from wine and other food. The medium is made of a general mineral medium, that is supplemented with vitamins and trace elements, glucose and formic acid as sources of carbon, acid base indicator and optionally with antibiotics. Here, the indicator is mostly bromocresol green.
On the blue selective medium, distributed by Millipore, we experience the discolouration around the Brettanomyces colonies, due to the acid they produce.
The solution (inventors: Loureiro Virgilio Borges and colleagues. “Culture medium for detection of Dekkera and Brettanomyces”) that was disclosed in the patent application, published under the number of EP1185686(A1) (it corresponds to the international publication no. WO2000073495A1), relates to a method and use of a general medium for identifying Dekkera and Brettanomyces yeasts, and determining their cell counts. According to the method, the following are added to the medium: nutrients; nonfermentable energy source, mainly ethanol; p-coumaric acid; acid base indicator, mainly bromocresol green; cycloheximide for blocking the growth of yeasts and antibiotics for blocking the growth of bacteria, chloramphenicol or oxytetracycline. The medium shows a distinctive discolouration as an effect of cultured (noble) colonies of Dekkera and Brettanomyces strains. The degree of discolouration changes depending on the growth pattern as an effect of decreasing pH. Furthermore, during culturing a distinctive, phenol-like aroma develops after a few days of incubation, which is easy to identify. The invention can be applied well in food industry.
In publication No. ES2268970(A1) Velazquez P. E. et al (“Yeasts detection culture medium comprises glucose mixed with buffer microorganism and bacterial growth inhibitors and e.g. a nitrogen source”) a medium suitable for the detection of yeasts has been taught, where the medium comprises glucose as carbon source, a calcium carbonate buffer, active agents capable of inhibiting the growth of different microorganisms and bacteria, nitrogen sources and a pH indicator which is actually neutral red. The medium is capable of detecting the Brettanomyces/Dekkera bruxellensis in foods and drinks. The identification is based on the acetic acid odour of the medium and on the appearance of transparent lines around the Dekkera/Brettanomyces yeast colonies present in the medium.
The Japanese patent application JP56106588 discloses a method for the production of a biological culture medium which contains methoxylated pectin and which is formed by admixing lactose (5 g), eosine Y (0.5 g), methylene blue (0.065 g), low methoxylated pectin (25 g) and deionized water (1 l) in the presence of agar-agar. The resulted mixture is sterilized, its pH is adjusted to 7.1 with sodium phosphate, and in a Petri dish a gel is poured from the resulted mixture. The culture medium is used for culturing yeasts, bacteria, microorganisms and fungi. The inventors do not have any knowledge about whether the medium is suitable for detecting yeast species, particularly Dekkera/Brettanomyces yeast colonies on the basis of colour change.
The above mentioned methods, that are based on culturing, can be conducted with minimal previous experience and they are cheap. In the case of the present methods, the identification is based mostly on the colour change of the indicator, for example, the organic acids produced by the Brettanomyces cause the acidification of the pH of the medium and thus the change of colour of the indicator. The selectivity of the methods were usually limited, therefore they were unable to distinguish certain wild yeasts from one another. The object of the invention is aimed to develop a medium and a selective culturing method which is more reliable and more easily estimable than previous ones and by the application of which, specific yeast colonies can be visually identified and unambiguously distinguished and separated from other microorganisms able to grow on the medium and which are relatively less problematic in the aspect of oenology.